Study of NPS1, a novel CDC gene of cerevisiae

NPSl基因是酿酒酵母的一个新的细胞分裂周期（CDC）基因，包含一个160 kd的细胞生长必需的核蛋白。我研究了NPS1基因在细胞周期中的功能，得到了以下结果。1)构建了nps1基因突变体，在蛋白激酶共识基序疑似atp结合位点的792位置有谷氨酸残基，而不是lysin残基（Lys792），发现Lys792对于nps1基因产物的体内功能是不可或缺的。2)通过对半乳糖培养基中仅能表达NPS1基因的条件致死菌株的表型研究，发现NPS1基因在酿酒酵母的g2期起作用，传递DNA复制完成的信号。3) NPS1基因产物定位于酵母核藏红花中，具有dna结合活性。4)用携带Nps1基因的重组杆状病毒转化SF9细胞，成功产生Nps1蛋白。目前正在进行转化SF9细胞的大规模培养和Nps1蛋白的纯化。5)为了鉴定与NPS1相关的基因，我进行了以下实验：1)分离出高剂量表达NPS1基因抑制生长的突变体；ii)筛选NPS1缺失致死性的高拷贝抑制因子；iii)筛选高剂量表达NPS1基因引起合成致死的基因。对上述实验获得的突变体和基因的分析正在进行中。

The NPSl gene is a novel cell division cycle (CDC) gene of Saccharomyces cerevisiae enconding a 160 kd nuclear protein essenntial for cell growth. I investigated the functions of NPS1 gene in cell cycle and obtained the following results.1) I constructed a mutant nps1 gene which has a glutamic acid residue at position 792 instead of lysin residue (Lys792) at a suspected ATP-binding site of protein kinase consennsus motief, and found that Lys792 is indispensable for in vivo function of NPS1 gene product.2) By investigating the phenotypes of conditional lethal strain which can express NPS1 gene only in galactose medium, I found that nps1 gene works in g2 phase of S.cerevisiae transducing the signal of completion of DNA replication.3) The NPS1 gene product was found to locate in nuclear saffold of S.cerevisiae and possess DNA-binding activity.4) I succeeded to produce the Nps1 protein in SF9 cell transformed with recombinant baculovirus carrying NPS1 gene. Large scaled culture of transformed SF9 cells and purification of Nps1 protein are now on going.5) In order to identify genes acting in relation to NPS1, I carried out the following experiments : i) isolate mutant whose growth is suppressed by the high dosage-expression of NPS1 gene ; ii) screen the high copy suppressor of lethality caused by NPS1 depletion ; iii) screen the gene which cause synthetic lethality with high-dosage expression of NPS1 gene. The analyzes of mutants and genes obtained by the above experiments are now on going.